Intracellular vesicular transport is essential for nearly all aspects of neutrophil physiology and defects in this mechanism leads to disease in humans. In neutrophils, vesicular trafficking is directly associated with the processes of exocytosis, phagocytosis, adhesion and chemotaxis; however, the molecular mechanisms that regulate mobilization of the different neutrophil secretory organelles during secretion, pathogen internalization or migration are incompletely understood. We have identified several key regulatory factors essential for the control of granule trafficking in neutrophils including the smll GTPase Rab27a that controls the mobilization and secretion of neutrophil granules in vitro and in vivo. This function is regulated by two Rab27a effectors, JFC1 and Munc13-4. Furthermore, we have recently identified previously unrecognized neutrophil secretory factors that interact with JFC1 and regulate cytoskeleton remodeling, vesicular trafficking and exocytosis and have pioneered the development of systems biology approaches to analyze vesicular dynamics and actin remodeling in granulocytes. In this application, we propose to use innovative analytical and quantitative methods to elucidate the mechanisms regulating vesicular transport associated with exocytosis, phagocytosis and chemotaxis in neutrophils. We also propose to use novel small molecule inhibitors of Rab27a-effector binding to interrogate mechanisms of vesicular transport and elucidate Rab27a-dependent processes in disease using an in vivo model of endotoxin-induced systemic inflammation. Since the toxic content of neutrophil granules is injurious to the host and neutrophil secretory proteins play a fundamental role in the damage to endothelium associated with endotoxemia and sepsis, these studies have important physiological significance and potential clinical applications in disease. We hypothesize that the differential regulation of vesicular transport by Rab27a and its effectors is an essential mechanism to determine specific neutrophil functions and responses to insult. We also propose that small molecule modulators of vesicular transport pathways will prevent some of the deleterious consequences of neutrophil secretion during systemic inflammation. To test our hypotheses we propose the following Specific Aims: 1) To define the mechanisms that regulate vesicular trafficking and actin remodeling during exocytosis in granulocytes and establish the role of the Rab27a effector JFC1 in this mechanism; 2) To establish the mechanisms regulated by Munc13-4 in vesicular transport and vesicular fusion associated with exocytosis, phagocytosis and bacterial killing; 3) To test the hypothesis that novel small molecule inhibitors of Rab27a-effector binding modulate neutrophil granule trafficking and exocytosis and attenuate inflammation. The results of the proposed research plan should uncover the molecular mechanisms regulating vesicular transport in neutrophils and lead to effective molecular strategies for the treatment of inflammatory diseases.